Composition and application thereof in preparation of medicine for preventing and treating psoriasis

Abstract

The invention relates to the technical field of biological medicine, in particular to a composition and application thereof in medicine for preventing and treating psoriasis. The composition comprises fumaric acid monoester and Al < 3 + >, and the research finds that the combination of aluminum ions and an Nrf2 agonist can relieve oxidative damage and regulate immune imbalance, plays a huge role in psoriasis treatment, solves the main problems of oxidative damage and immune imbalance in psoriasis pathogenesis, and has a good application prospect in treatment of psoriasis. Therefore, a new choice is provided for clinical treatment of psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a composition and its application in preparing a drug for preventing and treating psoriasis. Background Art

[0002] Psoriasis is an immune-mediated hyperproliferative inflammatory skin disease with red papules and plaques covered with white scales as the main clinical manifestations, and parakeratinization and hyperkeratinization of keratinocytes, abnormal proliferation, angiogenesis and inflammatory cell infiltration as the main histopathological features. The repeated attacks and difficulty in curing psoriasis not only seriously affect the physical and mental health of patients, but also increase the economic burden of patients' families. Therefore, in-depth exploration of the pathogenesis of psoriasis and finding safer, more effective and economical treatment options remain the research focus of the dermatology community. Recent studies have found that psoriasis is mainly an autoimmune, T cell-mediated disease. IL-23, IL-17 and TNF-α play a major role in the inflammatory response, leading to the occurrence and development of psoriasis lesions and their complications. The pathogenesis of psoriasis is mainly involved in Th1 and Th17 cytokines. In healthy individuals, these cytokines are regulated by regulatory T cells (Tregs). Tregs play a role in immune homeostasis, preventing the occurrence of autoimmune phenomena by suppressing immune responses. However, in psoriasis, the inhibitory function of Tregs is impaired, resulting in a change in the Th1 / Th17 balance. At the same time, studies have found that Nrf2 is reduced in skin lesions in an imiquimod (IMQ)-induced psoriasis mouse model. The accumulation of excessive ROS activates the NF-κB pathway and secretes the expression of proinflammatory cytokines IL-17, IL-23, and IL-1β.

[0003] Although there are many treatments available clinically to relieve psoriasis symptoms, unfortunately, no method has been reported to completely cure psoriasis. The main treatment method currently is to inhibit inflammatory responses through biological therapy, including TNF-α inhibitors, phototherapy, calcineurin inhibitors, retinoic acid drugs, and immunosuppressants. However, these methods are expensive and are accompanied by adverse reactions and drug dependence. Moreover, the high toxicity characteristics of the drugs make these therapies less than ideal.

[0004] Studies have found that Nrf2 plays a key role in the pathogenesis of psoriasis. Because Nrf2 plays an important role in redox homeostasis, cell protection, anti-inflammatory effects and skin barrier function, the scientific community has made considerable efforts to develop Nrf2-targeted drugs for the treatment of psoriasis. However, to date, only DMF (Nrf2 agonist containing α, β-unsaturated carbonyl groups) has become a successfully developed drug, which has been clinically proven to be a beneficial treatment option for psoriasis and multiple sclerosis. However, there are the following problems: drug dependence, single treatment method, high price, and in the process of clinical use, it was found that some patients did not respond to the above drugs, or some patients developed tolerance after using the above drugs for a period of time. Summary of the invention

[0005] In view of this, the present invention provides a composition for preventing and treating psoriasis, wherein fumaric acid monoester and Al 3+ It has a significant synergistic effect on the prevention and treatment of psoriasis.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] Composition comprising fumaric acid monoester and Al 3+ ;

[0008] The fumaric acid monoester includes monomethyl fumarate and / or monoethyl fumarate.

[0009] The present invention finds that monomethyl fumarate and Al 3+ They interact with each other, can significantly reduce oxidative damage and regulate immune imbalance, and play a significant synergistic role in the treatment of psoriasis.

[0010] In the present invention, Al 3+ The sources are aluminum chloride, alum (KAl(SO4)2·12H2O), aluminum sulfate, aluminum nitrate, etc., preferably aluminum chloride.

[0011] In some embodiments, the fumaric acid monoester and Al 3+ The molar ratio is (1~3):(1~3).

[0012] In some embodiments, the fumaric acid monoester is monomethyl fumarate, and the monomethyl fumarate and Al 3+ The molar ratio is 3:1, 1.5:1, 1:1, 1:1.5 or 1:3.

[0013] In some specific embodiments, the Al 3+ From aluminum chloride.

[0014] The present invention also provides application of the composition in preparing medicine for preventing and treating psoriasis.

[0015] In some embodiments, the prevention and treatment includes: reducing the level of pro-inflammatory factors, increasing the level of anti-inflammatory factors and / or reducing oxidative stress.

[0016] In some specific embodiments, the Th1 cytokines include IL-6, IL-1β, TNF-α and / or IL-17A; the Th2 cytokines include IL-4 and / or IL-10; and the reduction of oxidative stress includes: inhibiting NF-κB phosphorylation, reducing MDA levels, increasing GSH levels and / or reducing ROS levels.

[0017] In the application of the present invention, the effective concentration of the fumaric acid monoester in the composition is 100-800 μM.

[0018] In some specific embodiments, the effective concentration of the fumaric acid monoester in the composition is 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM or 800 μM.

[0019] The present invention also provides a drug for preventing and treating psoriasis, comprising the composition of the present invention.

[0020] In some embodiments, the drug may further include a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be any common type in the art, and the present invention does not make any special requirements therefor.

[0021] Those skilled in the art can select appropriate types of excipients according to needs and prepare the desired dosage forms according to conventional preparation methods in pharmacy, including but not limited to capsules, sustained-release preparations, powders, tablets, injections or pills.

[0022] The present invention also provides a method for preventing and treating psoriasis, which comprises: administering the composition of the present invention or the drug for preventing and treating psoriasis of the present invention.

[0023] The present invention provides a composition and its application in a drug for preventing and treating psoriasis. The composition comprises fumaric acid monoester and Al 3+ The present invention is the first to study and find that the combination of aluminum ions and Nrf2 agonists can reduce oxidative damage and regulate immune imbalance, play a huge role in the treatment of psoriasis, solve the main oxidative damage and immune imbalance in the cause of psoriasis, and thus provide a new option for the clinical treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 CCK8 was used to detect the viability of HaCaT cells after induction with different concentrations of MMF;

[0025] Figure 2Shows the effect of monomethyl fumarate combined with different metal ions; Real-time PCR was used to detect the expression of three indicators related to HaCaT cells: Th1 cytokine IL-6 (A), Th2 cytokine IL-10 (B) and antioxidant gene NQO1 (C); *p<0.05, **p<0.01;

[0026] Figure 3 The results showed that aluminum monomethyl fumarate can reduce the inflammatory storm and regulate the expression of Th2 cytokines; Real-time PCR was used to detect the expression of HaCaT cell-related inflammatory factors IL-6 (A) and IL-1β (B); the expression of Th2-related cytokines IL-4 (C) and IL-10 (D); p<0.05*, p<0.01**;

[0027] Figure 4 The results showed that aluminum monomethyl fumarate can activate the Nrf2 antioxidant signaling pathway; Real-time PCR was used to detect the expression of the key transcription factor Nrf2 (A), antioxidant enzyme HO-1 (B), and NQO1 (C) in HaCaT cells; D was used to detect the expression of Nrf2 protein by WB; the oxidative stress detection kit MDA and GSH were used to detect the expression of malondialdehyde (MDA) (E) and glutathione (GSH) (F); p<0.05*, p<0.01**;

[0028] Figure 5 The DCFH-DA fluorescent probe was used to detect the ROS in HaCaT cells of each group;

[0029] Figure 6 The results show that aluminum monomethyl fumarate can activate the nuclear entry of Nrf2 and reduce the level of NF-κB phosphorylation in HaCaT cells; A is the extraction of cytoplasmic and nuclear proteins using a nuclear cytoplasm separation kit, and WB detection of Nrf2 (Nuclear) and Nrf2 (Cytoplasm) protein expression; BC is the statistical graph of Figure A. D is the expression of NF-κB and phosphorylated NF-κB protein detected by WB. EF is the statistical graph of Figure D; p < 0.05*, p < 0.01**

[0030] Figure 7 The results showed that aluminum monomethyl fumarate was safer; A: The experiment was divided into 4 groups: saline group, MMF group, Al 3+ Group, MMF+Al 3+ Group (1:1); 1 ml of the corresponding drug was injected intraperitoneally into each group of Kunming mice, and their body weight was monitored and recorded every day; B: Body weight changes on the 4th day, p < 0.05*, p < 0.01*; C: The experiment was divided into 8 groups: saline group, MMF group, Al 3+ Group, MMF+Al 3+groups (1:1, 3:1, 1.5:1, 1:1.5, 1:3), each group of Kunming mice was intraperitoneally injected with 1 ml of the corresponding drug, and their body weights were monitored and recorded every day;

[0031] Figure 8 Figure 1 shows the establishment of psoriasis model; A is a schematic diagram of psoriasis modeling; B is the development of scales in the model group and the control group after 0, 3, 6, and 8 days of imiquimod infection in mice; C is a curve of daily body weight changes after imiquimod (IMQ) infection; D is the PASI score after IMQ application;

[0032] Fig. 9 The results show that aluminum monomethyl fumarate can relieve psoriasis; A represents the development of scales in the control group, model group and experimental group after 0, 3, 6 and 8 days of imiquimod infection in mice. BE is the PASI score of the back lesions of mice in each group: the scoring indicators include scales, erythema, and infiltration (each indicator score is 0-4 points), and the sum of the three is the total score (0-12 points); F is the PASI score on the sixth day, p < 0.05*, p < 0.01**

[0033] Fig.10 Shown HE and IHC detection of aluminum monomethyl fumarate to reduce the damage of the back skin of mice; A is the HE staining of the back skin tissue of mice in the control group, model group and experimental group; (B, C) After the HE results were obtained, the epidermal thickness and the infiltration of inflammatory cells were counted. The dotted box area is 0.01mm 2 D is an image of IHC of psoriatic skin; quantitative analysis includes (E) Ki67, (F) Ly6G, (G) IL-17, scale bar, 50 μm. p < 0.05*, p < 0.01**;

[0034] Fig.11 It shows that aluminum monomethyl fumarate can reduce the inflammatory storm and regulate the expression of Th2 cytokines;

[0035] Real-time PCR was used to detect the expression of inflammatory factors IL-1β (A), TNF-α (B), and IL-17A (C) in the lesion tissue of the psoriasis mouse model; the expression of Th2-related cytokines IL-4 (D) and IL-10 (E); p<0.05*, p<0.01**;

[0036] Fig.12 Monomethyl fumarate aluminum can reduce oxidative stress in the skin of psoriasis mice; WB was used to detect the expression of Nrf2, a key transcription factor of oxidative stress in the skin of psoriasis mice (A); Figure B is the gray value analysis of Figure A; The oxidative stress detection kit MDA and GSH were used to detect the expression of malondialdehyde MDA (C) and glutathione GSH (D). p<0.05*, p<0.01**. DETAILED DESCRIPTION

[0037] The present invention provides a composition and its use in a drug for preventing and treating psoriasis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] The present invention will be further described below in conjunction with embodiments:

[0040] Example 1

[0041] 1. Experimental Methods

[0042] (I) HaCaT cell recovery / passaging / culture and cryopreservation

[0043] (1) HaCaT cell recovery

[0044] ①Preparation before resuscitation: UV sterilization for 30 minutes.

[0045] ②Take out the frozen HaCaT cells and place them in a 37℃ water bath.

[0046] ③ After the cells thaw, centrifuge at 1000 rpm / min for 5 minutes to precipitate the cells and discard the freezing solution.

[0047] ④ Resuspend HaCaT cells in DMEM complete medium and inoculate them into culture dishes.

[0048] ⑤ Place the cells in a 37°C, 5% CO2 incubator and culture until the HaCaT cells are completely attached to the wall.

[0049] The cells were cultured continuously with new DMEM complete medium.

[0050] (2) HaCaT cell passage / culture

[0051] The growth of HaCaT cells was observed, and HaCaT cells were passaged when the cells grew to a density of about 90%.

[0052] ① After taking out the cells from the incubator, discard the old culture medium, wash them three times with sterile PBS, and add 0.25% pancreatic

[0053] The enzyme was allowed to soak the bottom of the culture dish.

[0054] ②Observe the state of HaCaT cells under an optical microscope. When HaCaT cells become round and bright, add DMEM

[0055] The digestion was terminated with complete medium.

[0056] ③The digested HaCaT cells were inoculated into a new culture dish at a ratio of 1:4 for culture.

[0057] (3) Cryopreservation of HaCaT cells

[0058] ① Configuration of freezing solution: 90% FBS + 10% DMSO.

[0059] ② The cells in good growth condition were digested with 0.25% trypsin to prepare a cell suspension.

[0060] ③ Add the freezing solution to the cell suspension to prepare a freezing solution suspension.

[0061] ④ Divide the cryopreservation liquid suspension into cryopreservation tubes, store them in a -80℃ refrigerator using a programmed cooling box, and transfer them to a liquid nitrogen tank for long-term storage after 24 hours.

[0062] (II) HaCaT cells were exposed to TNF-α and cell viability was detected by CCK-8 method

[0063] ① HaCaT was cultured and prepared into cell suspension after trypsin digestion.

[0064] ② 30% of the HaCaT cell suspension was inoculated into a 96-well plate for culture.

[0065] ③After 24 hours of TNF-α treatment, CCK8 working solution was prepared with DMEM complete medium at a ratio of 1:10 to form CCK8 application solution. 100 μL of CCK8 application solution was added to the 96-well plate and incubated at 37°C for 2 hours.

[0066] ⑤After incubation, use an enzyme reader to measure the absorbance (OD value) at a wavelength of 450nm.

[0067] (III) Detection of inflammatory indicators

[0068] (1) Cell culture

[0069] ① HaCaT cells were cultured, digested, and centrifuged to prepare a cell suspension.

[0070] ②HaCaT cells were counted and counted according to 1×10 4 / well were inoculated into 6-well plates.

[0071] ③ After HaCaT cells grow to the logarithmic growth phase, appropriate concentrations of MMF and Al 3+and MMF+Al 3 +Exposure to 20ng / ml TNF-α-induced inflammation model. Total RNA was extracted 24h later.

[0072] (2) Extraction of total RNA (Trizol method)

[0073] ① Add 750 μL of Trizol reagent to HaCaT cells and place on ice for 10 min. Then blow repeatedly to form a uniform suspension. Suck it into a 1.5 mL RNase free centrifuge tube, add 150 μL of chloroform, mix well and let stand for 5 min.

[0074] ②After standing, centrifuge at 4℃12000rpm / min for 15min, and use a pipette to remove 300μL of the upper liquid into another new 1.5mL RNase free centrifuge tube.

[0075] ③ Add 300 μL of isopropanol to a new RNase free centrifuge tube, mix well, and place in a -20℃ refrigerator for 20 min.

[0076] ④ After standing, centrifuge at 4℃ 12000rpm / min for 10min, discard the upper liquid in the centrifuge tube, and add pre-cooled 75% ethanol for washing.

[0077] ⑤ Continue centrifugation at 4℃ 12000rpm / min for 5min, repeat step ④ after the end, generally wash 2-3 times, place in a fume hood to dry after washing, wait until the white precipitate at the bottom of the centrifuge tube is completely transparent, add 20μL of DEPC water, which is the total RNA of HaCaT cells.

[0078] ⑥Nanodrop measures RNA concentration.

[0079] (3) Reverse transcription of RNA into cDNA (TaKaRa)

[0080] ① Select 20 μL of the transcription system in the TaKaRa reverse transcription kit for the experiment.

[0081] ②Calculate the amount of RNA required for transcription.

[0082] ③ Add 4 μL of 5x PrimeScript RT Master Mix and the required volume of RNA to a 200 μL EP tube, add water to make up the volume to 16 μL, shake and mix, and centrifuge to the bottom.

[0083] ④Set the reaction conditions on the PCR instrument: 37℃ 15min; 85℃ 5s; terminate at 4℃.

[0084] (4) Real-Time-PCR (TaKaRa)

[0085] ① Design primer sequences (Table 2).

[0086] ② Prepare sterile, enzyme-free pipette tips and 8-well tubes or 96-well plates for PCR. Prepare the following PCR reaction solution in a 0℃ metal bath (Table 1). Then, configure the mixing tubes according to the instructions based on the genes used in this experiment.

[0087] ③After the sample is loaded, observe whether there is any error in the sample loading, and then use a centrifuge to centrifuge the 8-tube or 96-well plate. Finally, set the reaction program of the PCR instrument according to the instructions (Table 3). After the setting is completed, load the sample into the machine to run the program.

[0088] ④Export data and calculate 2- ΔΔCT Make bar graphs and perform statistical analysis.

[0089] Table 1 RT-PCR system (20μL)

[0090]

[0091]

[0092] Table 2 Primer sequences

[0093]

[0094]

[0095] Table 3 Reaction procedure

[0096]

[0097] (5) Determination of oxidative stress

[0098] Malondialdehyde (MBA) detection

[0099] The experiment used Nanjing Jiancheng MDA (TBA method) detection kit.

[0100] ① Skin tissue and protein lysate were treated at a ratio of 1:9 to obtain protein homogenate.

[0101] ② Add samples according to the table below (Table 4) to a 1.5 mL centrifuge tube and repeat the sample test 3 times.

[0102] ③After adding the sample, place in a 95℃ water bath for 40 minutes.

[0103] ④After finishing, cool with running water to make the temperature drop slowly.

[0104] ⑤ Centrifuge at 4000rpm / min for 10min to separate the supernatant and the precipitate.

[0105] ⑥ Take 200 μL of supernatant from each centrifuge tube and place it in a 96-well plate, and measure the absorbance at 532 nm.

[0106] Calculation formula: MDA content in tissues or cells (nmol / mg protein) = (((measured OD-control OD) / (standard OD-blank OD)) × standard concentration) / test sample protein concentration (mg protein / mL)

[0107] Table 4 MDA operation steps

[0108]

[0109]

[0110] Trace reduced glutathione (GSH) detection

[0111] ① Sample pre-treatment:

[0112] Tissue samples: Skin tissue and protein lysis buffer were treated at a ratio of 1:9 to obtain protein homogenate.

[0113] ② After the sample is treated with reagent 1, add the sample according to the following table (Table 5).

[0114] Calculation formula for cells and tissues: Tissue and cell GSH content (μmol / L) = (((measured OD-blank OD)-(standard OD-blank OD))×20(μmol / L)×2) / sample concentration (mgprotein / mL).

[0115] Table 5 GSH operation steps

[0116]

[0117] (6) Western Blot experiment

[0118] ① Protein extraction:

[0119] Extraction of animal tissue protein: The skin tissue was quickly frozen with liquid nitrogen and then transferred to -80℃ for storage. During the experiment, the skin tissue was taken out and cut into pieces. Protein lysis buffer was added at a ratio of 1:9 and then the tissue was lysed using a tissue freezing grinder to extract protein.

[0120] Extraction of cell proteins: After treating HaCaT cells for 24 hours, protein lysis buffer was added to extract proteins.

[0121] ② Protein quantification (BCA method): Protein quantification was performed using the BCA assay method, using the Keygene Biotech BCA quantification kit, and the standard wells were loaded according to Table 6; then the wells to be tested were loaded. Each sample was repeated 3 times. After adding 200 μL of BCA working solution, the wells were placed in an ELISA reader, and after incubation for 30 minutes, the protein absorbance value (OD value) was measured at a wavelength of 562 nm.

[0122] ③ Protein denaturation: Quantify the protein according to the protein concentration, then add the protein loading buffer and denature it in a 100℃ metal bath for 10 minutes. After the denaturation, store it in a -20℃ refrigerator.

[0123] ④ Glue preparation: Prepare glue according to Table 7.

[0124] Table 6 Plotting of protein standard curve

[0125] Hole No. 0 1 2 3 4 5 6 7 Protein standard solution (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 Corresponding protein concentration (μg / μL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0126] Table 7 SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

[0127]

[0128] ⑤ Loading: 20μL of protein sample per well, add 2-4μL of different volumes of colored protein markers on both sides; during electrophoresis, first run the gel at 60V, and then adjust to 120V when the protein markers separate. Stop the electrophoresis when the bromophenol blue strip reaches the bottom of the gel.

[0129] ⑥ Transfer: First soak the PVDF membrane with methanol, make the gel and membrane into a sandwich structure according to the positive and negative electrodes, and finally use 200mA to transfer the membrane for 2h.

[0130] ⑦ Blocking: Place the membrane and 5% BSA solution in an antibody incubation box and block at room temperature for 1 hour.

[0131] ⑧Incubation of primary antibody: Take out the blocked membrane strips and cut the membrane according to the protein molecular weight, then add the corresponding antibodies and place the antibody incubation box on a shaker in a 4°C refrigerator overnight.

[0132] ⑨ Incubation of secondary antibody: After recovering the primary antibody, add PBST to wash the membrane, wash 3 times for 5 minutes each time. After washing, add the secondary antibody homologous to the primary antibody into the antibody incubation box and incubate at room temperature for 1 hour.

[0133] ⑩ECL luminescence: Use Bio-Rad's chemiluminescence system for protein development.

[0134] (IV) Establishment of animal model

[0135] (1) Experimental animals and drugs: 7-8 week old female Balbc mice, imiquimod ointment (Mingxinlidi)

[0136] (2) Hair removal of mice: One day before the experiment, the hair on the central area of ​​the mouse back was shaved with a mild depilatory cream. The hair removal area was approximately 2 cm × 2 cm.

[0137] (3) Psoriasis modeling: 5% imiquimod cream was applied at 50 mg / cm 2 Apply the dosage evenly to the hair removal area once a day for 8 consecutive days.

[0138] (V) HE staining

[0139] (1) Dehydration and embedding of skin tissue

[0140] ① Tissue fixation: The skin is fixed in 4% paraformaldehyde for 24 hours. After fixation, the skin tissue is placed in an embedding box and slowly rinsed with running water overnight.

[0141] ② Gradient alcohol dehydration: The tissue was dehydrated in the following order: 75% for 1h → 80% for 1.5h → 90% for 1h → 95% for 1h → 100% I for 20min → 100% II for 20min.

[0142] ③ Xylene is transparent: Xylene I 15min → Xylene II 15min.

[0143] ④ Wax immersion and embedding: After paraffin I1h → paraffin II1h, embed the skin tissue. After complete solidification, place it at 4℃ for long-term storage.

[0144] ⑤ Slicing and baking: Slice the skin tissue according to 5μm, then spread it in a constant temperature water bath at 40℃, place the slide with the spread skin tissue in an oven at 60℃ for 2h, and then store it at 4℃ for a long time.

[0145] (2) HE staining of skin

[0146] ① Dewaxing: Take out the preserved slides and bake them in a 60℃ oven for 40 minutes, then soak them in xylene I and xylene II for 15 minutes each.

[0147] ② Hydration: The slices were rehydrated according to the alcohol gradient of 100% II 3 min→100% I 3 min→95% 3 min→90% 3 min→85% 3 min→70% 3 min.

[0148] ③Hematoxylin staining: immerse in hematoxylin solution for 5 minutes.

[0149] ④Eosin staining: Place the slide in eosin staining solution for 3 minutes and rinse with running water for 1 minute.

[0150] ⑤ Dehydration: Dehydrate in alcohol in the following order: 70% for 1 min → 85% for 1 min → 90% for 1 min → 95% for 1 min → 100% I for 1 min → 100% II for 1 min.

[0151] ⑥ Transparency: Soak the slide in xylene I and xylene II for 3 minutes each to make it transparent.

[0152] ⑦ Sealing: Use neutral gum to seal the slides.

[0153] ⑧Photography: Take photos and observe using an optical microscope.

[0154] (3) Skin tissue immunohistochemistry (IHC)

[0155] ① Dewaxing: see HE staining of skin tissue.

[0156] ② Hydration: see HE staining of skin tissue.

[0157] ③Antigen repair: Select the appropriate antigen repair solution according to the antibody instructions and then perform repair.

[0158] ④ Blocking: Use an immunohistochemistry pen to draw a circle around the testicular tissue and completely soak the 3% hydrogen peroxide (H2O2) solution.

[0159] Cover the tissue and incubate in a dark box for 15 min.

[0160] ⑤ Blocking: After washing the excess peroxidase on the slide with PBS, gently shake off the excess water droplets on the slice.

[0161] After drying, add blocking solution to cover the tissue and place the slide in a dark box and incubate at room temperature for 1 hour.

[0162] ⑥ Primary antibody incubation: After blocking, add primary antibody to completely cover the tissue and incubate at 4°C overnight.

[0163] ⑦ Reaction enhancement solution: Take out the dark box the next day, place it at room temperature for 1 hour, wash it with PBS three times, and add appropriate amount of

[0164] The tissue was completely covered with the reaction enhancement solution and incubated at room temperature for 20 minutes.

[0165] ⑧ Secondary antibody incubation: After the primary antibody incubation is completed, wash the slides with PBS three times and add the enzyme corresponding to the primary antibody.

[0166] Incubate with secondary antibody at room temperature for 1 h.

[0167] ⑨DAB color development: Place a drop of prepared DAB color development solution in the center of the testicular tissue under a microscope. After observing the appearance of a specific yellow color in the skin tissue, immediately place the slide in water to terminate the color development.

[0168] ⑩The remaining steps are the same as HE staining of skin tissue.

[0169] (VI) Data processing and statistical analysis

[0170] All data in the experiment were repeated more than three times and analyzed by Graphpad prism 8 statistical software, and expressed as mean ± SD. The difference between the two groups was analyzed by t test; the comparison between multiple groups was performed by one-way ANOVA, * indicates P < 0.05, ** indicates P < 0.01.

[0171] 2. Experimental Results

[0172] (I) Screening of monomethyl fumarate combined with metal ions

[0173] First, we prepared monomethyl fumarate with a concentration of 50-1600 μM, exposed HaCaT cells, and performed CCK8 cell viability test. We found that the cells showed obvious death at a concentration of 800 μM. Therefore, we selected a concentration of 400 μM that did not affect cell viability for subsequent experiments ( Figure 1 ). We then prepared a fumarate metal ion mixture in a 1:1 concentration ratio: aluminum monomethyl fumarate, magnesium, calcium, manganese, and zinc. We selected the recognized method of modeling the psoriasis cell inflammation model, exposed HaCaT cells with 20ng / ml TNF-α, and added aluminum monomethyl fumarate, magnesium, calcium, manganese, and zinc. We used qPCR to detect three indicators: Th1 type cytokine IL-6, Th2 type cytokine IL-10, and antioxidant gene NQO1. The results showed that aluminum monomethyl fumarate could significantly reduce the expression of IL-6, while increasing the expression of IL-10 and NQO1, and only aluminum monomethyl fumarate had the most obvious effect ( Figure 2 ).

[0174] (II) Aluminum monomethyl fumarate can reduce inflammatory cytokine storm and oxidative stress in HaCat cells induced by TNF-α

[0175] (1) Aluminum monomethyl fumarate can reduce the inflammatory storm of HaCaT cells and regulate the expression of Th2 cytokines

[0176] In order to explore the effect of aluminum monomethyl fumarate on the HaCaT inflammatory cell model, we set up Control, TNF-α, MMF, Al 3+ and MMF+Al 3+The gene expression levels of Th1 cytokines IL-6, IL-1β, and Th2 cytokines IL-4, IL-10 were detected. The results showed that TNF-α could cause a significant increase in IL-6 and IL-1β genes and a significant decrease in IL-4 and IL-10 genes in HaCaT cells. The MMF group could reduce the expression of Th1 cytokines, Al 3+ The combined group could significantly increase the expression of Th2 cytokines, while the combined group could both reduce the expression of Th1 cytokines and increase the expression of Th2 cytokines ( Figure 3 ).

[0177] (2) Aluminum monomethyl fumarate can activate the Nrf2 antioxidant signaling pathway

[0178] In order to detect the effect of monomethyl fumarate aluminum on the Nrf2 antioxidant signaling pathway, we first detected the gene levels of Nrf2, HO-1, and NQO1, and found that monomethyl fumarate aluminum can activate the levels of Nrf2's downstream genes HO-1 and NQO1, but does not affect the gene level of Nrf2. Therefore, we detected the protein expression level of Nrf2 and found that both monomethyl fumarate and monomethyl fumarate aluminum can upregulate the protein level of Nrf2. At the same time, the MDA and GSH kits were used to detect the levels of MDA malondialdehyde and GSH glutathione. We found that monomethyl fumarate aluminum can further reduce the level of MDA and increase the level of GSH compared with monomethyl fumarate ( Figure 4 Finally, the DCFH-DA fluorescent probe was used to detect the level of endogenous ROS. Compared with monomethyl fumarate, aluminum monomethyl fumarate could further reduce the level of ROS in HaCaT cells ( Figure 5 ).

[0179] (3) Aluminum monomethyl fumarate can activate the nuclear translocation of Nrf2 and reduce the level of NF-KB phosphorylation in HaCaT cells

[0180] In order to explore the mechanism of the antioxidant effect of monomethyl fumarate combined with aluminum ions, we used a nuclear cytoplasm separation kit to extract nuclear and cytoplasmic proteins and detect the nuclear entry of Nrf2. The results showed that monomethyl fumarate combined with aluminum ions can further lead to the nuclear entry of Nrf2. At the same time, we detected the levels of NF-κB and phosphorylated NF-κB. The results showed that monomethyl fumarate aluminum can further inhibit the level of phosphorylated NF-κB compared with monomethyl fumarate ( Figure 6 ). In summary, our study showed that monomethyl fumarate combined with aluminum ions can activate the nuclear translocation of Nrf2 and inhibit the phosphorylation of NF-κB, thereby exerting anti-inflammatory and antioxidant effects.

[0181] (III) Safety Experiment

[0182] We then verified the safety of the combination. 3+ Group and MMF+Al 3+ Groups (numbered 4 to 8), see Table 8, the drugs were injected into KM mice (intraperitoneal injection). The safety of the drugs was observed by observing the weight loss of KM mice.

[0183] Table 8 Experimental groups and administration methods

[0184]

[0185] The results showed that compared with the single action group, the combination group of the present invention showed less weight loss, among which the combination groups with a compound ratio of 1:1 and 1.5:1 had the least weight loss ( Figure 7 A~C).

[0186] (IV) Establishment of psoriasis model

[0187] We used imiquimod to continuously apply to the backs of depilated mice for 8 days and recorded their weight and back photos. We found that the weight of mice in the model group was significantly reduced, and the PASI score (international standard psoriasis scoring standard) also showed that the psoriasis model was successfully established ( Figure 8 ).

[0188] (V) Treatment of animal models of psoriasis with monomethyl fumarate and monomethyl fumarate aluminum

[0189] Monomethyl fumarate was used in combination with aluminum, and a blank group, a model group, and a treatment group (monomethyl fumarate group, aluminum ion group, and monomethyl fumarate combined with aluminum group) were established. The psoriasis model group was intraperitoneally injected with the drug on days 1, 4, and 7. The weight changes of the mice were recorded every day, and the changes in the back skin of the mice were recorded using a digital camera on days 1, 4, and 7. On day 9, the mice were euthanized, and the back skin was fixed in 4% paraformaldehyde after observing the back blood vessels.

[0190] First, on the sixth day, the three treatment groups showed significant therapeutic effects compared with the model group. Among them, the therapeutic effect of the monomethyl fumarate combined with aluminum group was significantly better than that of the monomethyl fumarate group and the aluminum ion group ( Fig. 9 ).in, Fig. 9 The specific data of the total score of E on the sixth day are shown in Table 9.

[0191] Table 9

[0192]

[0193] Among them, the scale score includes the scale area and number.

[0194] The back skin of psoriasis mice was collected for HE staining. The results showed that the epidermis of the model group mice was significantly thickened and the infiltration of inflammatory cells increased significantly, while the three treatment groups were able to reverse this phenomenon, among which the combined group had the best effect ( Fig.10 AC). Finally, epidermal cell proliferation marker (Ki67), neutrophil infiltration marker (Ly6G) and inflammatory factor (IL-17) were detected by immunohistochemistry (IHC). The results showed that the combined group could significantly reduce the expression of Ki67, Ly6G and IL-17, and the effect was the best compared with the single treatment group ( Fig.10 DG).

[0195] (VI) Study on the anti-inflammatory and antioxidant effects of aluminum monomethyl fumarate

[0196] (1) Aluminum monomethyl fumarate can reduce the inflammatory storm in the skin lesions of psoriasis mice and regulate the expression of Th2 cytokines

[0197] In order to explore the effect of aluminum monomethyl fumarate on the psoriasis mouse model, we detected the gene expression levels of Th1 cytokines IL1β, TNF-α and IL-17A, and Th2 cytokines IL-4 and IL-10. The results showed that IMQ could cause a significant increase in the IL-1β, TNF-α and IL-17A genes in the epidermis of psoriasis mice and a significant decrease in the IL-4 and IL-10 genes. The combined group could significantly reduce the expression of Th1 cytokines and increase the expression of Th2 cytokines, and the effect was more significant than that of the single action group ( Fig.11 ).

[0198] (2) Aluminum monomethyl fumarate can activate the Nrf2 antioxidant signaling pathway

[0199] In order to detect the effect of aluminum monomethyl fumarate on the Nrf2 antioxidant signaling pathway in the skin of psoriasis mice, we detected the expression of Nrf2 protein and found that aluminum monomethyl fumarate could upregulate the level of Nrf2 protein. At the same time, MDA malondialdehyde and GSH glutathione levels were detected using MDA and GSH kits. We found that aluminum monomethyl fumarate could further reduce the level of MDA and increase the level of GSH compared with monomethyl fumarate ( Fig.12 ).

[0200] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that Including fumaric acid monoester and Al 3+ ; The fumaric acid monoester includes monomethyl fumarate and / or monoethyl fumarate.

2. The composition according to claim 1, characterized in that The fumaric acid monoester and Al 3+ The molar ratio is (1~3):(1~3).

3. The composition according to claim 2, characterized in that: The fumaric acid monoester is monomethyl fumarate, and the monomethyl fumarate and Al 3+ The molar ratio is 1:

1.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing and treating psoriasis.

5. The use according to claim 4, characterized in that: The prevention and treatment include: reducing the level of pro-inflammatory factors, increasing the level of anti-inflammatory factors and / or reducing oxidative stress.

6. The use according to claim 5, characterized in that: The Th1 cytokines include IL-6, IL-1β, TNF-α and / or IL-17A; The Th2 cytokines include IL-4 and / or IL-10; The reducing of oxidative stress includes: inhibiting NF-κB phosphorylation, reducing MDA level, increasing GSH level and / or reducing ROS level.

7. A drug for preventing and treating psoriasis, characterized in that: The invention comprises the composition according to any one of claims 1 to 3.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 7 or 8, characterized in that Its dosage forms include capsules, sustained-release preparations, powders, tablets, injections or pills.

Images